Neuroscience Letters, 140 (1992) 185 188

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© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

NSL 08690

Nerve growth factor regulation of choline acetyltransferase gene expression in rat embryo basal forebrain cultures M a t t h e w V. Lorenzi a, Beat Knusel b, Franz Hefti b and William L. Strauss a ~Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, MiamL FL 33101 (USA) and hAndrus Gerontology Center, University of Southern California, Los Angeles, CA 90089 (USA) (Received 28 January 1992; Accepted 6 March 1992)

Key words: Cholinergic neuron; Acetylcholine synthesis; Neurotrophic factor Nerve growth factor (NGF) increases the activity of choline acetyltransferase (CHAT), the synthetic enzyme for acetylcholine, in rat basal forebrain neurons both in vivo and in vitro. In poly(A) ÷ RNA isolated from cultures prepared from the embryonic (El5) rat basal forebrain, radiolabeled probes from the human ChAT gene detected a 3,700 nt and a less abundant 2,300 nt transcript. After growth in the presence of NGF, the abundance of both mRNAs was increased approximately twofold, parallelling the increase in ChAT enzyme activity. In vivo, the human ChAT probes detected a single 3,700 nt form of ChAT mRNA in both embryonic and adult rat basal forebrain. These results suggest that the NGF-mediated increase in ChAT activity in basal forebrain cultures is regulated at the transcriptional level.

Nerve growth factor (NGF) exerts a pronounced trophic effect on the cholinergic neurons of the basal forebrain during development and throughout adult life [11]. Intraventricular injections of NGF increase choline acetyltransferase (CHAT, EC 2.3.1.6, acetyl-CoA:cholineO-acetyltransferase) activity in the basal forebrains of neonatal rats and support the survival of these neurons after fimbrial transections in the adult [7, 10]. Several laboratories have demonstrated that the effects of NGF on ChAT activity and cholinergic neuron survival can be reproduced in cultures of rat embryo forebrain [8, 9, 15]. Some of the actions of NGF on central cholinergic neurons are comparable to those produced by cholinergic differentiation factor (CDF) in the periphery. It recently was reported that neonatal rat superior cervical ganglion neurons in culture express a 2,700 nt mRNA that hybridizes to a rat ChAT cDNA probe. Growth of these cultures in the presence of CDF, which increases ChAT enzyme activity, induces a larger 4,000 nt ChAT mRNA, but not the 2,700 nt transcript expressed in control cultures [16]. To determine whether NGF increases ChAT activity in central cholinergic neurons through a similar change in transcription, we have used exon-specific

Correspondence: M.V. Lorenzi, Department of Molecular and Cellular Pharmacology, P.O. Box 016189, University of Miami, School of Medicine, Miami, FL 33101, USA. Fax: (1) (305) 548-4555.

probes from the human ChAT gene to examine rat ChAT mRNA in vivo and in vitro. Cultures were prepared from the basal forebrains of embryonic age 15 (El 5) fetal rats as described previously [8]. The dissected area contained cholinergic neurons belonging to the medial septal nucleus and the diagonal band of Broca. Neurons were grown in a modified L-15 medium supplemented with 5% horse serum and 0.5% fetal bovine serum (Gibco). Culture medium was exchanged every other day. After 3 days growth in culture, human recombinant NGF (Genentech) was administered at 100 ng/ml to treated cultures. After 10 days total RNA was isolated from the cultures as described below. ChAT enzymatic activity in basal forebrain cultures was measured by the method of Fonnum [6]. Protein was determined using the bicinchoninic acid protein assay (Pierce). Total RNA was isolated from culture monolayers and from tissue samples by the methods of Falvaloro et al. [5] and Chirgwin et al. [3], respectively. Briefly, monolayers of cultured cells were lysed in 0.14 sodium chloride, 0.0015 M magnesium chloride, 0.010 M Tris (pH 8.6) containing 1000 units/ml RNasin (Promega) and 0.5% nonidet P-40. After shearing chromosomal DNA by 3 passes through a 21 gauge needle, proteins were digested with proteinase K (200/~g/ml, Boehringer Mannheim) and extracted with phenol:chloroform. Total RNA was isolated from embryonic and adult rat brain by homog-

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Fig. 1. ChAT m R N A abundance in the adult rat CNS. Top: 10/2g of total RNA from the indicated brain regions were resolved on a 1% agarose gel containing 0.7% formaldehyde, transferred to a Nitroplus 2000 membrane and hybridized to [3zp]-labeled pChAT1.2, an 800 bp genomic DNA probe containing an exon of the human ChAT gene. The autoradiogram was exposed at -70°C for 3 days with intensifying screens. Bottom: histogram of the relative abundance of ChAT m R N A in the adult rat CNS normalized to y-actin mRNA. Total R N A samples were also hybridized to a human 7-actin cDNA probe. The abundance of the 3,700 nt transcript was normalized to y-actin m R N A expression within the selected brain regions.

enization in 4 M guanidine isothiocyanate followed by ultracentrifugation through a 5.7 M cesium chloride cushion. Poly(A)+ RNA was enriched by batchwise purification using the PolyATtract kit (Promega) following the manufacturer's instructions. The concentration of RNA from both preparations was estimated spectrophotometrically at an optical density of 260 nm. Total RNA (10/lg) or poly(A) + RNA (1/lg) was fractionated on 1% agarose gels containing 0.7% formaldehyde and blotted onto Nitroplus 2000 membranes by standard methods. The 800 bp subclone of the human ChAT gene ([17], pChAT1.2) and a 900 bp human 7actin cDNA [4] were prepared for radiolabeling by linearization with HindIII and EcoRI, respectively. DNA probes were labeled with [32p]dCTP (New England Nuclear) using the random priming method to a specific activity of approximately 1 x 10 9 cpm//2g DNA. Following overnight hybridization, filters were washed at 55°C in 1 x SSC, 0.1% SDS and exposed to X-AR film at -70°C with an intensifying screen for the indicated times. Autographic images were quantified using a Zeineh scanning densitometer. The 800 bp subclone of the human ChAT gene (pChAT1.2) used to detect rat ChAT mRNA has been described elsewhere [17]. To determine the conditions under which this probe would specifically detect rat ChAT mRNA, pChAT1.2 was hybridized to total RNA extracted from regions of the adult rat CNS. In agreement with a previous report [1], pChAT1.2 detected a

single 3,700 nt mRNA in the adult rat CNS (Fig. 1). The abundance of this transcript was normalized to the expression of 7-actin mRNA within the selected brain regions (Fig. 1). As expected, ChAT mRNA was most abundant in the septum and basal forebrain. Low levels of ChAT mRNA were observed in the striatum and cortex. Although ChAT mRNA was not detected in the hippocampus or kidney, high levels of 7-actin mRNA were observed (data not shown). To evaluate the effects of NGF on ChAT mRNA abundance, pChAT1.2 was hybridized to poly(A) + RNA isolated from control and NGF-treated rat basal forebrain cultures. After 10 days in culture, cells grown in control medium expressed the 3,700 nt mRNA (Fig. 2). Furthermore, the pChAT1.2 probe detected a faint signal corresponding to a 2,300 nt species o f m R N A (Fig. 2, lane 1). Both the 3,700 and 2,300 nt transcripts also were detected by a second protein coding exon from the human ChAT gene, pChAT5 (Lorenzi and Strauss, unpublished results). Growth in the presence of NGF (100 ng/ ml) increased the abundance (normalized to the abundance of y-actin mRNA in each sample) of the 3,700 nt mRNA approximately twofold (Fig. 2, lane 2). The intensity of the signal for the 2,300 nt transcript also was enhanced. In a parallel set of cultures NGF administration resulted in an approximately twofold increase in the enzymatic activity of ChAT (control cultures: 1.2 nmol acetylcholine synthesized per min per mg protein; NGFtreated cultures: 2.5 nmol acetylcholine synthesized per min per mg protein). The presence of the 2,300 nt transcript in cultures of embryonic cells, but not in adult tissue, suggested that this might be an embryonic ChAT mRNA. Alternatively, the abundance of the 2,300 nt transcript in the total RNA sample used in the adult rat brain analysis may be below the limits of detection of Northern analysis. To examine these possibilities further, pChAT1.2 was hybridized to poly(A)+ RNA isolated directly from both E15 and adult rat basal forebrain. A single 3,700 nt species of mRNA hybridized to pChATt.2 in both adult (Fig. 2, lane 4) and embryonic day 15 basal forebrain (Fig. 2, lane 3). No other hybridization was observed in longer exposures of autoradiograms (data not shown). NGF supports the survival and neurite growth of embryonic rat forebrain cholinergic neurons in culture [8, 9]. Furthermore, NGF increases ChAT activity and the intensity of immunocytochemical staining obtained with anti-ChAT antibodies [8, 14], suggesting that NGF upregulates the expression of the ChAT gene. The finding that NGF-treated cultures contain higher levels of ChAT mRNA confirms this hypothesis and suggests that the regulation of ChAT activity by NGF occurs at the transcriptional level. This conclusion is supported by re-

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Fig. 2. Developmental and neurotrophic effects on ChAT mRNA expression. A: the DNA probe prepared from pChAT1.2 was hybridized to 1/.tg of poly(A)÷ RNA isolated from control cultures (lane 1), NGFtreated cultures (lane 2), embryonic day 15 (El5) rat basal forebrain (lane 3) or adult basal forebrain (lane 4). Lanes 1-4 of A were exposed to X-ray film at -70°C for 24 h. B: the expression of y-actin mRNA in one microgram of poly(A)÷ RNA for the RNA preparations described in A. Lanes from the autoradiogram were exposed for 15 h (lanes 1 and 2) or 24 h (lanes 3 and 4) at -70°C.

cent results indicating that, in PC 12 cells, N G F exposure increases the activity o f the C h A T gene p r o m o t e r [13]. The stimulatory effect o f N G F on C h A T gene transcription is not limited to early development, since Cavicchioli et al. [2] f o u n d that intraventricular infusions o f N G F in adult rats increases septal C h A T m R N A levels measured by an assay based on the polymerase chain reaction. In vivo, a 3,700 nt C h A T m R N A is expressed by embryonic (E 15) and adult tissue. A species o f identical size was present in our cultures, as well as a less a b u n d a n t 2,300 nt species. Recently, multiple species o f C h A T m R N A have been observed in Drosophila [18], rats [12, 16] and h u m a n s (Lorenzi and Strauss, unpublished ob-

servation). In the adult rat testis, 3.5 kb and 1.3 kb are detected by a full length rat C h A T c D N A [12]. A m o r e detailed analysis o f these transcripts has revealed that the two rat testes m R N A s share the first 800 bp o f coding sequence but differ at their 3' ends [12]. The 3,700 nt and 2,300 nt m R N A s which we detect in basal forebrain cultures hybridize with h u m a n C h A T exons at the 5' and 3' ends o f this same region suggesting that the two m R N A s we detect m a y be the neuronal counterparts o f the C h A T m R N A s observed in rat testes. Two m R N A s o f similar size (4,000 and 2,700 nt) which hybridize to a full length rat C h A T c D N A have been reported by N a w a et al. [16] in cultures o f neonatal rat superior cervical ganglion (SCG) neurons g r o w n in the presence o f CDF. In control cultures o f S C G neurons, which express C h A T enzyme activity, only the 2,700 nt transcript was observed. C D F appears to induce the appearance o f the larger C h A T m R N A in S C G n e u r o n cultures [16]. In contrast, N G F increases the a b u n d a n c e o f b o t h m R N A s which we detect in basal forebrain cultures. It is possible that the 2,300 nt m R N A m a y be an embryonic f o r m o f C h A T m R N A which is regulated in vivo by one or m o r e additional n e u r o t r o p h i c factors. These results suggest that the regulation o f C h A T gene expression during development is likely to be m o r e complicated than had previously been appreciated. This w o r k was supported by N I H G r a n t NS23430 to W.L.S. and by N S F G r a n t BNS-9021255 to F . H . M . V . L . is a predoctoral fellow o f the Pharmaceutical M a n u f a c turers' Association F o u n d a t i o n . N G F was the generous gift o f Genentech.

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